A combination of selective synthesis, degradation and transport produces a non-random phospholipid distribution across the plasma membrane of most eukaryotic cells. The long-term objectives of our research program are to understand the mechanisms by which this asymmetric phospholipid distribution is established and its significance to cellular function. Preliminary experiments demonstrated that in the yeast, S. cerevisiae, inward-directed transport (flip) of phosphatidyicholine (PtdCho), as reported by a short chain, fluorescent-labeled PtdCho (NBD-PC), is coupled to the plasma membrane, proton-electrochemical gradient and is down-regulated by nutrient deprivation and by activated drug resistance transcription factors. A classical mutagenesis approach identified a loss of function mutation in a gene that reduces NBD-PC flip by >90% and dramatically increases resistance to the toxic lysophospholipid analogue, ET-18-O-CH3. This gene is predicted to encode a membrane protein with two transmembrane domains and has no identifiable functional motifs. Although no molecular function has been identified, two additional homologues exist in the S. cerevisiae genome as well as in a wide range of metazoans including humans. The specific aims of this proposal are to test the hypothesis that the three yeast genes encode plasma membrane-localized, inward-directed, phospholipid transporters (flippases) that are regulated in response to growth state and toxic stress. Specifically these genes will be characterized by classic molecular, genetic, and biochemical analyses to determine the relationship of their expression to in vivo function, their cellular location, specific domains essential to function, interactions with other proteins, response to nutrient deprivation and drug resistance factors, and their in vitro flippase activity. In addition to providing a better understanding of the role of phospholipid membrane dynamics to cell function, these studies will likely identify the mechanism of internalization of ET-18-O-CH3, and other ether lipid drugs, that have anti-fungal. anti-protozoal, and anti-neoplastic activity. Understanding the mechanism by which these drugs are internalized may lead to the discovery of new drugs of this class that are internalized more efficiently and are less susceptible to the development of resistance.